Main
Issues and Achievements of the Laboratory

Last revision on 09/04/97

Our laboratory is studying
Archaea and hyperthermophilic Bacteria living at
temperatures close to 100°C. Our goal is to understand
how can these organisms "function" at the limits of life,
and to establish whether they originated as a result of
primary or secondary adaptation. We are particularly
interested in the DNA World which has probably set up at the
time of the Archaea, Bacteria and Eucaryotes divergence.
These studies have led us to focus on the important issues
of origin and evolution of Genomes.

We are
working on DNA topology and DNA topoisomerases in
Archaea and Hyperthermophilic (HT) Bacteria. We
have shown that HT Archaeal plasmids are either
relaxed or positively supercoiled, meaning that
their DNA have an excess of topological links as
compared to mesophilic plasmids. Moreover, we have
discovered that HT Archaea possess a completely new
family of type II topoisomerases, which led us to
identify in silico a protein which
could be responsible for the initiation of
crossing-over in Eucaryotes. We are interested in
HT plasmids as markers for DNA topology studies,
templates for in vitro HT replication
systems, and cloning vectors

This issue is
the basis of our commitment to an European
biotechnology project (BIOTECH) in collaboration
with the laboratories of Drs. D. Prieur in Roscoff
(France) and R. Garrett in Copenhagen (Denmark):
the aim is to setup the conditions for the culture
of HT Archaea in liquid and solid media, on Petri
dishes; to setup and validate efficient
transformation methods, as well as to isolate
mutants resistant to growth inhibitors, in order to
obtain genetic markers which will be ultimately
inserted in cloning/expression vectors for
hyperthermophiles. We have already discovered
several new plasmids in HT Archaea of the
Thermococcales genus. One of them has been fully
sequenced and his replication mechanism has been
identified as being of the rolling circle type.

We have
identified duplicated (paralogous) gene families in
E. coli . This work and other
phylogenetic studies undertaken in our laboratory
have led us to settle the importance of gene
duplications before the Eucaryotes/Procaryotes
separation. Finally, we have shown that published
results, dealing with rooting of the universal tree
in the bacterial branch, are questionable.

DNA
World at High Temperature

DNA under HT conditions
in vitro (P. Forterre, E. Marguet).
We have shown that a closed circular DNA is not denatured
under HT conditions, at last until 107°C ; however,
it is rapidly degraded by a mechanism involving
depurination followed by the phosphodiester bond breakage
(15).
This thermodegradation is independent of DNA topology and
is inhibited by high concentrations of monovalent salts.
(15).Thus,
Reverse Gyrase from HT do not seem to be necessary for
DNA protection against overall denaturation or
degradation.

In vivo DNA in HT
Archaea and extreme halophiles(F. Charbonnier, F.
Mojica, P. Lopez-Garcia, E. Marguet, D. Gadelle, coll.
with D. Prieur, W. Zillig and F. Rodriguez -Valera).
We have isolated pGT5, the first plasmid from a HT
Archaea, Pyrococcus abyssi (3)
. Moreover we have shown that the plasmid is relaxed at
optimal growth temperature (95°C) (4).This
conclusion can be applied to other HT plasmids
(13),
as isolation of new plasmids from Thermococcales and
Sulfolobales, shows that they are all either relaxed, or
positively supercoiled. (25).
On the contrary, plasmids isolated from halophilic
archaea are negatively supercoiled (13,16,17).
Thus, DNA isolated from HT archaea exhibits a linking
number (Lk) excess compared to mesophilic DNA. This
excess (presumably due to Reverse Gyrase) could maintain
DNA in a structural conformation close to it's state in
mesophiles. Accordingly, we have shown that the linking
number increases during heat shock and decreases during
cold shock in HT. Furthermore, Lk raises upon temperature
increase in halophilic and HT archaea (17)
as well as in mesophiles. Gyrase and reverse gyrase
activities could have appeared during procaryotic
evolution in order to allow their adaptation to a very
broad temperature range (0 - 110°C)
(25).

HT DNA topoisomerases
(A. Bergerat, O. Guipaud, D. Gadelle, B. Labedan, coll.
with M. Duguet and F. Robb).
We are collaborating with M. Duguet's laboratory for
several years now on Topoisomerases I, and more
specifically on HT Reverse Gyrase. (7,
8).
As this lab is carrying out a thorough study of
topoisomerases I, we have concentrated our efforts on
topo II. Do HT bacteria bearing reverse gyrase also
possess a gyrase? We have cloned genes coding for 2
subunits of topo II from HT bacteria T. maritima .
Subunits corresponding to GyrB (gyrase) and/or ParE (topo
IV) have been fully sequenced, allowing us to draw a
distance tree based on the 25 already known GyrB/ParE
sequences. As opposed to the 16s rRNA tree, T.
maritima doesn't branch deeply on the tree, but
rather near B. subtilis , a result also derived
from other proteic phylogenetic trees. Poor resolution of
branches doesn't allow us to tell whether T.
maritima 's topo II is a gyrase or a topo IV.
Purification of the enzyme is under way in our
laboratory. We have purified to homogeneity and
characterized topoisomerases II from HT archaea
Sulfolobus shibatae (18)
and P. furiosus . Both enzymes are heterotetramers
constituted by 45 and 60 kd proteins for A and B subunits
respectively, and resistant to usual topo II inhibitors.
ATP is binding to B subunit. Peptide sequences have been
obtained from S. shibatae A and B subunits,
allowing us to clone and fully sequence the genes, which
are contiguous and correspond well to purified
proteins.

Studies on pGT5
Plasmid (S. Marsin, N. Rollet, Y. Zivanovic, coll.
with D. Prieur).
pGT5 plasmid (3.4 kb) has been completely sequenced
(27).
The sequence shows evidences for a rolling circle type of
replication: a gene coding for an endonuclease-ligase
(Rep) related protein, found in the pC194 plasmid family,
as well as two potential, single-stranded(sso) and
double-stranded (dso), replication origins (ori). The
putative sso resembles ori sequences recognized by
bacterial primosome. We have demonstrated the existence
of a single stranded replicative intermediate of pGT5 in
P. abyssi , which constitutes a signature of the
rolling circle mechanism (27).

First archaeal genes
related to DNA repair, chromosome structure and cell
division [ A. Bouyoub, E. Gerard (coll. with G.
Barbier et J. Querellou), C. Elie].
We have searched for DNA polymerases genes in three
Thermococcales strains (IFREMER collection). A DNA
polymerase gene, from the GE8 strain, containing an
intein has been isolated. An gene involved in the SOS
response, anE. coli dinF homologue, has been
identified in strain 700 (24).
In bacteria, recA or lexA can be found upstream of the
dinF gene. We have cloned and sequenced a gene upstream
of dinF in strain 700, but it appears to be homologous to
the E. coli minD gene, involved in cellular
division. In the course of this work, we have also
isolated the purA gene from strain 707, which is now
tested as a potential genetic marker (see below). Still
during our DNA polymerase search, we have discovered in
Sulfolobus acidocaldarius an homologue of
eucaryotic ATPases belonging to the SMC family
(Structural Maintenance of the Chromosome) and of yeast
RAD50 gene. These are motor, dumbbell shaped, proteins
bearing the coiled-coiled motif, which play a key role in
chromosome condensation or recombinaison.

Genetic
Tools for Hyperthermophilic Archaea

This work is headed by Y. Zivanovic in coll. with
Prieur and Garrett in the frame of an european program.
We have shown that pGT5 bearing an insert in between the
two main open reading frames of the plasmid can be
transferred and replicated in Pyrococcus cells
(26).
However, it is unstable at the moment, which could be due
to it's replication mode and/or to the lack of efficient
selection marker in this type of construct.

Discovery of new plasmids in Thermococcales,
characterization of new strains (N. Rollet, P.
Lopez-Garcia).
In about twenty Thermococcales strains already studied,
six different plasmids have been discovered. Their sizes
range from 3,5 to 25 kb. Some of them have been detected
by PFGE analysis of total genomes. Moreover, genomic
sizes of several HT archaea have been determined (1.7 to
3.9 Mb).

Isolation of the purA gene from Pyrococcus and
potential purA mutants . (Y. Zivanovic )
Culture conditions for anaerobic HT archaea have been
established in the laboratory (e.g. setup of an anaerobic
chamber). We have isolated, by colony plating at
95°C, several P. abyssi mutants
resistant to 6-methyl-purine in presence of hypoxanthine,
potentially defective in the purA gene. Mutant and wild
type purA genes have been cloned and are currently under
way of sequencing. Various constructs have been made by
merging archaeal pGT5 and bacterial Litmus plasmids along
with resistant purA genes.

Origin
and Evolution of Genomes

Identification of duplicated (paralogous) genes
families in E. coli.(B. Labedan, coll. with M.
Riley).
Analysis of the full set of known E. coli genes
has permitted to show that 52% of genes are paralogous
(originating from the same ancestor by duplication)
(22,
23,
54). Various levels of paralogy have been identified,
allowing us to define ancestry relationships among groups
of genes. 747 known E. coli sequences could
originate from only 92 ancestral sequences
(23,54).

Evidences for gene duplications before the
eucaryotes/procaryotes divergence. (N. Benachenhou,
P. Forterre, B. Labedan).
Phylogenetic analysis of GDHs [we have cloned the
S. shibatae GDH gene (14)],
DNA polymerases (2,
11),
DNA topoisomerases I and II (11),
OTCases and ATCases (coll. with Glansdorff) has
demonstrated that phylogenetic trees thus obtained are
not species trees. Their topology imply that some gene
duplication took place before the eucaryotes/ procaryotes
divergence (9).
This result has led us to criticize rooting of the
universal tree of life in the bacterial branch, a work
based on ATPases analysis (6,
10),
this tree being itself spoilt by paralogy. The case of an
horizontal gene transfer has been studied in coll. with
J.Guespin (19).These
analysis could be linked in some instances [GDH
(14),
purA] with the search for HT signatures. In
particular, numerous deletions in the purA protein from
Pyrococcus could lead to a more compact structure
of the protein.

Origin of HT, use of the clastidic method to
solve the universal tree rooting problem. (P.
Forterre, C. Elie).
Molecular analysis of HT, in particular the study of the
reverse gyrase gene, has led us to question the existence
of a link between the hot origin of life and the outbreak
of HT (20,
28).
We have proposed a novel hypothesis to explain why the
common ancestor of archaea and bacteria was an HT
(21,
iv)
: the outbreak of the "procaryotic phenotype" by
thermoreduction. According to this hypothesis, rooting of
the universal tree falls in the eucaryotic branch.
However, a bacterial rooting of the tree has been
proposed based on elongation factors and ILeu tRNA
synthetases phylogenetic analysis. Nevertheless, by
applying a strict cladistic analysis to these protein
sequence alignments, we have shown that the bacterial
rooting of the tree is not robust. (6).
In order to determine whether bacteria and archaea were
specifically linked, we tried to establish whether they
possess the same type of replication origin (ori). We
sequenced 6 kb of Haloferax volcanii chromosome
upstream of the gyrB gene (this region is conserved among
bacteria and contain very often dnaA and oriC). We have
identified an orf which shows week similarity with dnaN,
a gene close to OriC in many bacteria, but we didn't find
any homologue to dnaA.